Faststart universal sybr green pcr master

Total RNAs were extracted from different tissues using RNA Isolation Reagent (Invitrogen, Carlsbad, CA) and reversely transcribed to produce cDNA (Fermentas, EU). Relative expression of messenger RNA (mRNA) species was determined by Real Time PCR with Faststart Universal SYBR Green PCR Master (Roche Diagnostics, USA) by ABI7300. The primer sequences were shown in Additional file 1: Table S1. Relative mRNA levels were normalized with β-actin, and results were expressed as fold amplification.

Trizol reagent (GK20008, GLPBIO, USA) was used to extract total RNAs. High-Capacity cDNA Reverse Transcription Kit (Cat#43744967; Thermo Fisher Scientific, Waltham, USA) was used to synthesize cDNA. qRT-PCR analysis was performed using 1 μL cDNA, 1 μL forward primer, 1 μL reverse primer, and FastStart Universal SYBR Green PCR Master (Cat#04913914001; Roche, Switzerland) by a 7500 Fast Real-Time instrument (Roche, Switzerland). The expression levels of the target genes were normalized to GAPDH gene. The primer pair sequences used in the present study are listed in Supplementary Table 1.

A total of 38 candidate hub genes were selected for RT-qPCR analysis to examine their expression profiles. Primer pairs of candidate hub genes were designed by Beacon Designer 8.0 (Table S9). The buds of YT93-159 and ROC22 at 0 d, 1 d, 2 d, and 5 d time points post-S. spontaneum inoculation were selected as materials. GAPDH was used as the internal reference gene [95 (link)]. The gene expression analysis of candidate hub genes was carried out with three independent biological replicates for each sample [96 (link)]. RT-qPCR was performed on ABI QuantStudio^TM 3 system (Thermo Fisher Scientific, Waltham, MA, USA) using the SYBR-green dye method following a thermal cycling program of “50 °C, 2 min; 95 °C, 10 min; 40 cycles of (95 °C, 15 s; 60 °C, 1 min)”. The total reaction volume was 25 µL, including 12.5 µL FastStart Universal SYBR Green PCR Master (Roche, Shanghai, China), 0.5 µL of each primer (10 µM), 1.0 µL template (10× diluted cDNA liquid), and 10.5 µL ddH₂O. Expression levels of selected hub genes were calculated using the 2^−∆∆CT method [97 (link)]. Histograms were graphed by GraphPad Prism 6. Significance (p < 0.05) and standard error (SE) were determined by the Duncan’s new multiple range test.